1. Field of the Invention
The present invention is directed to the field of immunotherapy and more particularly to a glycoconjugate, a composition and vaccine comprising the same and to the use thereof for enhancing the immune response and notably in cancer therapy and in therapeutic of infection caused by pathogenic agent against whom a humoral or a cellular immune response is necessary. The invention relates also to a diagnosis kit and a method for diagnosis of cancer.
2. Prior Art/Relevant Literature
As a result of aberrant glycosylation, cancer-associated carbohydrate antigens are exposed at the surface of tumor cells whereas they are hidden in normal cells (Ref. 1). Recent advances in immunology and in the identification of tumor specific antigens have renewed the interest for the development of cancer vaccines, and these exposed glycosidic B-cell epitopes have been considered as attractive targets for immunotherapy named “Active Specific Immunotherapy” (ASI) by Longenecker (Ref. 2). This approach involves immunization with a defined antigen to elicit a specific immune response to that antigen and could represent an alternative to the conventional cancer therapies.
Among the large number of known tumor markers, the Tn (a-GalNAc-Ser/Thr). the T* (b-Gal-(1→3)-a-GalNAc-Ser/Thr) and the sialosyl-Tn (a-NeuAc-(2→6)-a-GalNAc-Ser/Thr) antigens have been extensively studied since they are expressed on mucin-type glycoproteins by the majority of adenocarcinomas (Ref. 3), Indeed, several studies have shown some protection against tumors after immunization with these glycosidic antigens, in experimenital or clinical studies. These tumour associated carbohydrates are relevant markers for cancer diagnostic and prognosis (Ref. 34). Using desialyated red blood cells, which are rich in T and Tn determinants, Springer observed a long-term effective protection against recurrence of human breast carcinoma (Ref. 3c, Ref. 4). An other group investigated the potential of ASI with desialylated ovine submaxillary mucin (d-OSM), which contains high density of the Tn epitope; their studies showed that this antigen provided a good protection and a long-term survival in mice with mammary carcinoma (Ref. 5). Partially-d-OSM also gave efficient protection against human colon carcinoma (Ref. 6). Ratcliffe et al. were the first to use a synthetic tumor-associated antigen, a T antigen-protein conjugate, to stimulate an efficient immune response in rabbits (Ref. 7). Thereafter Longenecker extensively studied similar synthetic carbohydrate hapten conjugates and found that they induce an increased survival of mice grafted with mammary carcinoma cells (Ref. 8, and of patients with ovarian cancers (Ref. 9). Similar studies of the same group have further shown an increased protection of patients suffering from breast cancer (Ref. 10) or melanoma (Ref. 11) after respective administration of sialosyl Tn- or the GM2 ganglioside-protein conjugates. On the other hand, Toyokuni et al. generated an anti-tumor antibody response in mice after immunization with a Tn antigen coupled either to OSA (Ovine Serum Albumin) or to a synthetic lipopeptide (Ref. 12). This last result was interesting since it was the first example of a small synthetic carbohydrate antigen that generates an immune response against a tumor associated carbohydrate antigen, without the use of a macromolecular carrier or adjuvants.
These studies suggested that carbohydrate antigens are appropriate candidates for anti-tumor vaccine development. However, carbohydrate antigens do not possess T-cell epilope and therefore induce only weak T cell-independent antibody response. Several approaches have been explored to increase the immunogenicity of such carbohydrates. The use of biological material which expresses clusters of antigens on a protein backbone (like desialylated red blood cells or OSM) is a possibility. But the most widely used approach is to conjugate the carbohydrate to a carrier protein, such as Bovine serum albumin (BSA) or Keyhole limpet hemocyanin (KLH).
Although these immunogens have shown some promise, protein carriers display major disadvantages. The grafted epitope represents only a small part of the total conjugate and it is distributed at random on the carrier surface. Therefore, immune responses to the carrier molecule may result in a low level of the desired antibodies as compared to the total amount of antibodies produced. Moreover, these conjugates present ambiguity in both composition and structure and they do not always induce reproducible immune response. Recent advances in the total synthesis of oligosaccharides expressed by tumour cells (Ref. 35. Ref. 36) open new possibilities for such achievement. However, haptenic molecules such as carbohydrates require their association in more complex structures to stimulate immune responses. The use of traditional protein conjugates raises the problem of hapten-specific suppression (Ref. 37. Ref. 38), and their poorly defined chemical composition and structure may limit their efficacy.
Until now, as for chemically defined structures, dendrimeric poly-lysine backbones, which will be described in more detail later in the present specification, have been widely used for presenting peptides (Ref. 14). However, to our knowledge, there is only one preliminary attempt of their utilisation for presenting carbohydrates to the immune system (Ref. 16). This latter reference teaches the synthesis of three sialylated multiple antigen peptides having tetanus toxin T-cell epitopes. However a response against the T cell epitope only was obtained, but not against the B cell epitope. A similar strategy was also recently published (Ref. 17) where the authors coupled mixtures of natural polysaccharides obtained from Streptococcus and Saccharomyces to a Multiple Antigenic Peptidic system.
Thus, there still exist a need for a new conjugate circumventing the drawbacks mentioned above of the prior art constructions which has a chemically defined structure, is capable of stimulatiny both the antibody response and the T response when administered in a human or animal body while avoiding undesired immune responses.